(A) Fluorescence spectra of mlCNBD-FRET at 430 nm excitation before (black) and after addition of 10 μM cAMP (grey). (B) Schematic representation of the structural changes evoked by cAMP upon binding, FRET becomes smaller, indicating that cerulean and citrine move further apart. (C) Structural changes occurring after cAMP binding. The cAMP-free structure is shown in blue, the cAMP-bound structure is shown in green. Distances are presented in Ångstrom. (D) Binding of cAMP to mlCNBD-FRET (black) determined by fluorescence spectroscopy. Representative experiments showing an increase in the baseline-corrected cerulean/citrine emission ratio (△F) of mlCNBD-FRET (430 nm excitation) after cAMP binding. Data have been fitted using a single binding-site model (red line) (Cukkemane et al., 2007). As a control, mlCNBD-FRET-R307Q (red dots) has been used. Measurements have been performed using 1 μM protein. (E) Representative experiments showing an increase of △F of mlCNBD-FRET (430 nm excitation) after cGMP binding. Data has been fitted (see (D), red line). Measurements have been performed using 1 μM protein. (F) Normalized fluorescence spectra of mlCNBD-FRET (430 nm excitation) at different pH conditions. Spectra were normalized to the cerulean emission at 471 nm. (G) Normalized FRET (430 nm excitation, 529 nm emission) at different pH values. Data have been taken from measurements shown in (F) and are presented as mean ± S.D.; n = 3. (H) Kinetics of cAMP binding to mlCNBD-FRET measured using the stopped-flow technique. Different cAMP concentrations (in µM: 0.5, 1, 1.6, 2.5, 5, and 10) were mixed with the purified mlCNBD-FRET protein (2.5 μM) and the change in FRET was measured over time. Solid lines represent a global fit of a one-step model (see materials and methods) with the following parameters: kon = 2.6 *107 M-1s-1 and koff = 12.8 s-1.